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Cancer Epidemiology Biomarkers & Prevention, Vol 6, Issue 7 531-536, Copyright © 1997 by American Association for Cancer Research

ARTICLES

S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy

H Konishi, G Steinbach, NH Terry, K Fujita, JJ Lee, A Ruifrok, D Spaulding, PM Lynch, JA Dubin, M Andreeff, AM Goodacre, T Hattori and WN Hittelman
Department of Gastrointestinal Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.

Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified-starved, and refed rats were labeled by histone H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P < 0.001) discriminated the proliferative differences between control, modified-starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.


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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
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Copyright © 1997 by the American Association for Cancer Research.